Inflammatory bowel disease (IBD) refers to a spectrum of genetic and phenotypically complex disorders that are thought to result from dysregulated immune responses to commensal microbes in genetically susceptible hosts. In IBD, the intestinal epithelium is characterized by increased permeability both in active disease and remission states, and increased permeability has been associated with elevated risk of relapse, yet the precise mechanisms remain undefined. Proteins associated with inflammation and bacteria can increase permeability, however, evidence from relatives of IBD probands suggests a genetic association as well. Progress in understanding the genetic relationship with intestinal permeability is impeded by a lack of scientific approaches to address the issue. This Ancillary R01 proposal engages the objectives of The NIDDK IBD Genetics Consortium (IBDGC) and benefits from its repository of lymphoblastoid cell lines (LCLs) generated from IBD patients and its contribution to the efforts that have identified 163 loci associated with IBD. We have developed an experimental system to approach genetic associations with intestinal permeability. Using SNPs associated with intestinal epithelium and permeability, we generated a weighted risk scoring system based on each SNP's association with IBD to selected LCLs to reprogram to form induced pluripotent stem cells (IPSCs), and to subsequently direct these IPSCs to form three-dimensional human intestinal organoids (HIOs). These HIOs contain all intestinal epithelial subtypes, possess adherens junctions and have polarized tight junctions. These HIOs will be used to study the functional relationships between SNPs and intestinal permeability. We will also use linked biospecimen tissue to assess alterations/mislocalizations of tight/adherens junctions. Lastly, patients from whom the HIOs were derived will be recalled to assess intestinal permeability - allowing us to meet a secondary objective of the NIDDK IBDGC - the rapid application of findings to patient care. The HIO system allows us to assess functional outcomes of these SNPs under defined conditions while the linked biospecimens and in vivo permeability tests allow corroboration of these outcomes in an ex vivo/in vivo setting. We hypothesize that a high risk score predisposes towards increased intestinal permeability under either basal or a subset of inflammatory conditions as compared to a low risk score and that this difference will be reproduced in a patient setting. We will approach our hypothesis through the following specific aims: 1) Determine if a high risk score is associated with alterations in the composition of HIOs; 2) Determine if a high risk score is associated with changes in the permeability of HIOs; and 3) Determine if the high risk score associated changes in tight/adherens junction composition and structure in HIOs, is reflected in small bowel tissues and in vivo permeability measurements in genotyped IBD patients. Results will establish if there is a genetic influence on intestinal permeability in IBD and delineate SNPs responsible for increased permeability.